DDRT-PCR and silver staining

D. KIM dkim at NMSU.Edu
Thu Oct 15 11:45:05 EST 1998


Hello:

I have used the  Promega Silver SEquence staining kit to stain DDRT gels,
and then recovered the DNA from the stained bands for PCR amplification.
There was no problem with this method, except that each band contains a
variety of DNA species, only one of which is significant to the
differential display.  Several strategies have been published to address
this problem.  In any case, amplification of bands is not a problem.
Re-amplification of silver-stained SSCP bands was reported in
BioTechniques, I think.

Silver Sequence kit uses the rapid silver stain method of B. Bassam and ?.
Caetano-Annoless, which was published in Analytical Biochemistry in the
early 1990's or late 1980's.  The method is simple and can be done using
reagents readily available.  Pay extra money for a better grade of silver
nitrate, and use reasonably fresh sodium thiosulfate, for cleaner
staining.

Daniel Kim
dkim at nmsu.edu

KwangChul Oh <ohk at ucs.orst.edu> wrote:
: Hi..

: I am planing to use silver staining kit to detect DDRT-PCR product on
: TBE-urea gel. The PCR-product will be reamplified to make probe. I am
: concerned whether fixative containing acid such as acetic acid, TCA and
: sulphosalicylic acid may modify or destroy the bases. Do I have to use
: isotope instead of silver staining?

: Thank you for help

: -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

: KwangChul Oh

: ohk at bcc.orst.edu





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